大肠杆菌O_(157):H_7 rfbE基因和fliC基因的克隆与序列分析

Cloning and Sequencing of rfbE gene and fliC gene in E.coli O_(157):H_7

根据已发表的E.coliO_(157):H_7EDL933株的rfbE基因和fliC基因,设计了两对引物,分别对两个O_(157):H_7菌株的rfbE基因和一个菌株的fliC基因进行PCR,并将PCR产物克隆于pMD18-T载体质粒。测序结果表明,获得到了与理论相符的582bprfbE基因片段和802bpfliC基因片段。一株菌的rfbE基因存在无意义突变(两个)。而另一株菌fliC基因有一个有意义突变(aac→gac)

To clone and analysize rfbE and fliC genes of E.coli O157：H7 , two primers were designed according to the sequence of rfbE gene and fliC gene of E.coli O157：H7 EDL933 strain.The part fragment of rfbE gene of two E.coli O157：H7 ,882364 and 021210 strain, were amplified by polymerase chain reaction （PCR） respectively. Both PCR products were cloned into pMD-18 vector and identified by BamH I ,HindlII and EcoRI and two positive recombinants containing rfbE gene were selected and sequenced.With the same method another positive recombinant containing fliC gene from 021210 strain was analysized. The result suggested homology of the rfbE gene between 882364 and 021210 strain was 99% for nucleotide sequence . While the homology of the fliC gene compared with GENBANK sequence （AF228487）was 99%.